The structure and function of the antibody related to the present invention will be first explained. Namely, such structure and function are described in details in Kabat et al.: Sequences of proteins of immunological interest, 4th Ed., 1989, NIH, U.S.A. as well as Roitt et al.: Immunology, 2nd Ed., 1989, Gower Medical Publishing, U.S.A. & U.K.
The antibody (immunoglobulin) is an antigen-specific glycoprotein as produced by B lymphocytes when a subject is sensitized with an antigen which is a foreign substance to the subject. As the human immunoglobulin, there are known 5 classes, i.e., IgG, IMP, IgA, IgD and IgE. In case of IgG, it has two light chains (L chains) of polypeptide having a molecular weight of about 25,000 and two heavy chains (H chains) of polypeptide having a molecular weight of about 51,000. Between H-L chains and between H-H chains, there is usually present a disulfide bond connecting two chains. An amino acid sequence consisting of about 100 amino acids at N terminal of each of the H and L chains is antigen-specific and represents an antigen-binding site. This part is called a variable (V) region. Subsequent amino acid sequence consisting of 400 amino acids in H chain or 150 amino acids in L chain is called a constant (C) region which is identical among all immunoglobulins belonging to a Ig Class such as IgG or IgM Class, or those belonging to a subclass such as igG.sub.1 or IgG.sub.2. It is known that human IgG may have C.kappa. or C.lambda. in L chain and C.gamma.1, C.gamma.2, C.gamma.3 or C.gamma.4 in H chain. L and H chains having one of these identified partial structures are called .kappa., .lambda., .gamma.1, .gamma.2, .gamma.3 and .gamma.4 chain, respectively.
H and L chain contain "domain structures". For instance, H chain is composed of VH, CH1, CH2, CH3 domains and hinge regions connecting CH1 and CH2 domains.
The variable region comprises four framework regions in which relatively conservative amino acid sequences are retained among various antibodies and three CDRs which are relatively variable in the amino acid sequence among different antibodies. In one molecule of an antibody which comprises two H chains and two L chains, there ar present six CDRs originated from VH and VL regions, which take steric configurations closely approached one another to form an antigen binding site.
The summary of the structure and function of the antibody is as above.
The monoclonal antibody (hereinafter, referred to as "MAb") is widely used for diagnosis and therapy in the medical field and as reagents, affinity column materials, etc. in the industrial field. The mouse MAb is readily obtained by the mouse/mouse hybridoma method.
For preparation of a human MAb which is more valuable for human therapy that a mouse MAb, various improvements have been proposed but any reliable method for establishing a producible cell line with good reproducibility and high efficiency has not been established. Because of this reason, human MAb as clinically usable is quite restricted. Also, the production of a human antibody to an antigen originated from a human being is generally impossible except any special case.
In case of a mouse, MAb specific to various antigens including antigens of human origin can be easily obtained but on administration to a human being, a problem of antigenicity occurs. Some attempts have been made to produce an antibody lowered in antigenicity to a human body from mouse MAb. Specifically, attempts are directed to the production of a humanized antibody wherein only a CDR, which is said to form an antigen-binding site, in the variable region of mouse MAb, is left and all the other regions are replaced by the human ones.
For instance, in the method as disclosed in EP-A-87302620, the CDR of mouse MAb is transplanted into the human MAb V region by the use of site specific mutation with a long oligonucleotide. As an example of obtaining a humanized antibody as explained above, there is known an attempt for humanization of rat MAb Campath-1 recognizing CDw52 antigen on human T calls (EP-A-89301291).
As one of mouse MAbs for which humanization would be effective, there is known anti-human IL-2 receptor antibody B-B10 (Japanese Patent Publication (Unexamined) No. 2-13371). This antibody is antagonistic to the binding of IL-2 to the IL-2 receptor on human T-cells and inhibits the IL-2 dependent growth of activated T-cells. It also inhibits the human mixed lymphocyte reaction. Accordingly, said MAb is useful for treatment and prevention of the diseases caused by graft-versus-host reaction or host-versus-graft reaction, prevention of rejection on the transplantation of bone marrow, kidney, heart, lung, pancreas, skin, liver, etc., therapy of T-cell dependent allergy or autoimmune diseases (E.g., myocarditis, diabetes mellitus, myasthenia gravis, lupus erythematosus, Crohn disease, multiple sclerosis, AIDS, Meningitis, Arthritis) and therapy of tumors expressing IL-2 receptor such as T-cell leukemia.
In fact, the administration of B-B10 on the graft-versus-host reaction as produced after the bone marrow transplantation or the preventive administration of B-B10 on the rejection of the liver transplantation produces a certain effect (Blood, Vol. 75, 1017 (1990); Lancet, Vol. 335, 1596 (1990)).
On the practical therapy, however, the administration of B-B10 is carried out only for a short period of time, because of a concern to the antigenicity of mouse MAb. Also, there is a clinical example where an antibody to mouse MAb was found from a patient to whom mouse MAb was administered. The administration over a long period of time is thus quite difficult from the practical viewpoint. As understood from this, the administration of B-B10 is limited due to the fact that it is a kind of mouse MAb and the therapeutic effect is also restricted.
In order to solve the above problem, it is necessary to decrease the antigenicity originated from the mouse antibody by humanization. As to humanized antibodies, there are present some other examples in addition to those as hereinabove mentioned. For instance, an anti-Tac antibody is humanized by transplantation of nine amino acid residues on the framework in addition to CDR, and as the result of the humanization, the activity is lowered to 1/3 (Proc. Natl. Acad. Sci. USA Vol. 86, 10029 (1989)). Also, the humanized antibodies to the gB glycoprotein and gD glycoprotein of herpes simplex virus are transplanted respectively with two amino acids residues and eight amino acid residues on the framework in addition to CDR, and their activities are respectively 1/2 and 1 in comparison with mouse MAb (Proc. Natl. Acad. Sci. USA, Vol. 88, 2869 (1991)). Further, the humanized antibody to the human CD4 is transplanted with one amino acid residue in addition to CDR, and its activity is 1/3 in comparison with mouse MAb (Proc. Natl. Acad. Sci. USA, Vol. 88, 4181 (1991)).
As understood from the above, it is necessary for obtaining a humanized antibody having an activity similar to mouse MAb to transplant not only CDR but also an amino acid residue(s) which would afford an important influence on the antigen-antibody binding in the framework of mouse MAb. However, such amino acid residue(s) are different depending upon the kind of the antibody; in fact, the examples as above recited shown that some amino acid residues are common to them and some others are not. It is thus required to determine the necessary amino acid residue(s) on each antibody. Like the case of the humanized antibody to the gD glycoprotein of herpes simplex virus, such an approach as leaving the amino acid sequence on the framework expected to participate in the antigen-antibody binding as in mouse MAb. In this case, however, the same amino acid residue as in mouse MAb increase so that the antigenicity of mouse MAb is increased.
When the framework of mouse MAb contains one or more amino acid residues which rarely exist in human antibodies, they are, in principle, concerted to other amino acid residues highly common to human antibodies. Thus, sixteen amino acid residues on the framework have been substituted in this case. As understood from the above, it is necessary for obtaining a humanized antibody to identify amino acid residue(s) on the framework which appear essential for retaining the activity and to transplant them to the human antibody together with CDR. However, it is very difficult to pre-determine such essential amino acid residue(s). Accordingly, transplantation of all of the amino acid residues on the framework, which appear possibly involved in antigen-binding activity, is desirable in preparing humanized antibody. However, the humanized antibody thus prepared is destined to include many amino acid residues derived from mouse antibody, and therefore, antigenicity of the humanized antibody may inevitably be high.